Clostridium difficile causes disease within the gastrointestinal tract, usually following an alteration of the intestinal microecology. The pathogenic role of toxigenic C. difficile in antibiotic-associated pseudomembranous colitis in humans is well established. Disease symptoms vary with the source of the pathogen, antibiotic exposure, toxin production, and patient age.
C. difficile produces two toxins, designated A and B. The toxins are implicated in the etilology of the disease. They can be separated by ion exchange chromatography and identified according to their elution behavior from DEAE ion exchange. Toxin A elutes at 0.15M NaCl, and may be further purified by isoelectric precipitation. Toxin B elutes at 0.3M NaCl, and is further purified by affinity chromatography. Toxin A is primarily an enterotoxin with slight cytotoxic activity, whereas toxin B is a potent cytotoxin. Toxin A causes extensive damage to the gut mucosa, as well as accumulation of fluid in the intestinal tract It is believed that the primary event in the mechanism of C. difficile infection involves the.specific binding to receptors on the intestinal cell surface. However, "receptor" as used herein means simply the chemical structure which binds toxin A without regard to whether or not that structure is in fact implicated in the disease-causing process.
C. difficile-associated intestinal disease has been reported in infants, and in adults in the absence of antibiotic therapy. Moreover, C. difficile is one of the most common bacterial enteropathogens found in stool specimens in hospitals. The organism has been reported to be one of the most commonly detected bacterial pathogens of enteric disease. In the United States, the disease now exceeds the total number of illnesses caused by C. botulinum, C. tetani and C. perfringens.
C. difficile causes pseudomembranous colitis in humans as a result of the elimination of the normal flora of the colon by antibiotic usage and growth of this toxin-producing bacterium. The disease usually occurs in hospitalized patients where it causes a massive diahhrea with extensive inflammation of the colon. Without proper treatment, the patient is likely to die. Treatment is based on a proper diagnosis which is accomplished by establishing the presence of the toxin and demonstrating the characteristic lesions in the colon.
Recent success in the purification of toxins A and B has stimulated a great deal of interest in their biological properties. Further study may lead to an understanding of how the toxins cause disease. However, existing methods for purifying these proteins are costly, require skilled personnel, and demand several days to obtain pure toxin.
The existing methods for detecting pathogenic C. difficile are inadequate. One such test involves the culture of human feces, which requires specialized facilities for a long period of incubation. This test suffers from interference by non-pathogenic C. difficile strains, namely strains not producing toxin. The test is costly, time-consuming, and can only be performed in larger, well-equipped hospitals or in private laboratories.
A more innovation in the detection of pathogenic C. difficile involves the use of specific antisera to toxin A coated onto latex beads. Lyerly, et al, A.J. Clin. Microbiol. 17:72-78 (1983). The resulting immobilized antibody agglutinates soluble toxin A. The agglutination of the large latex beads can be easily seen, indicating the presence of toxin A in the sample However, the method is expensive since it relies upon costly monospecific antibodies to toxin A.
What is needed is a simple, rapid and inexpensive method for obtaining highly purified toxin A, and a simple, rapid and inexpensive test for detecting the presence of toxin A which can be performed in any hospital laboratory.